Fiber scaffolds for enhancing cell proliferation in cell culture

ABSTRACT

A substrate for culturing cells that includes at least one fiber scaffold adapted to be contained within a disposable or non-disposable bioreactor, wherein the fiber scaffold further includes polymer fibers that have been created by electrospinning, and wherein the orientation of the fibers in the scaffold relative to one another is generally parallel, random, or both.

BACKGROUND OF THE INVENTION

The described invention relates in general to systems and devices foruse in cell culture, and more specifically to a cell culture system andsubstrate that includes one or more polymer scaffolds adapted to becontained within a bioreactor, wherein the fiber scaffolds furtherinclude polymer fibers that have been created by electrospinning, andwherein the orientation of the fibers in the scaffold relative to oneanother is generally parallel, random, or both.

Certain types of mammalian cells require attachment to a substrate sothat they may adequately proliferate and undergo normal cellularfunction. These cells typically include a variety of stem or progenitorcells such as, for example, mesenchymal stem cells (MSC), and are ofinterest for a variety of clinical applications and therapies. However,the number of viable cells required for a typical therapeutic dose canbe millions or billions per patient. Additionally, stem cells easilydifferentiate into other undesirable cell types while being expanded invitro, thereby reducing the efficiency of the expansion process andcreating a major problem for stem cell suppliers. Accordingly, there isa significant need for commercially available technologies that arecapable of greatly expanding a relatively small number of stem orprogenitor cells into a much larger population of such cells whilemaintaining the pluripotency thereof.

Current commercially used cell expansion processes typically involvelarge reusable stainless steel or glass bioreactors that must be cleanedand disinfected between batches or disposable, single-use bioreactorsthat resemble plastic bags. Stem cells are added to these bioreactorswith appropriate media and reagents for promoting cell growth and thenthe bioreactors are closely monitored until a desired concentration ofcells is achieved. For adherent cells, porous beads made frompolystyrene and other polymers are added into the growth media and cellmixture to allow the cells to attach and grow normally. However, oncethe desired concentration of cells is achieved it is very difficult toremove all of the cells from the porous beads. This results in a lowefficiency of usable cells for the desired end application. Therefore,there is an ongoing need for a cell culture system that permits desiredcell growth and proliferation and that allows cultured cells to beharvested efficiently and in large numbers.

SUMMARY OF THE INVENTION

The following provides a summary of certain exemplary embodiments of thepresent invention. This summary is not an extensive overview and is notintended to identify key or critical aspects or elements of the presentinvention or to delineate its scope.

In accordance with one aspect of the present invention, a substrate forculturing cells is provided. This substrate includes at least one fiberscaffold adapted to be contained within a bioreactor. The fiber scaffoldfurther includes polymer fibers that have been created byelectrospinning and the orientation of the fibers in the scaffoldrelative to one another is generally parallel, random, or both.

In accordance with another aspect of the present invention, a system forculturing cells is provided. This system includes at least one fiberscaffold adapted to be contained within a bioreactor, and a bioreactor.The fiber scaffold further includes polymer fibers that have beencreated by electrospinning and the orientation of the fibers in thescaffold relative to one another is generally parallel, random, or both.The bioreactor may be disposable or permanent (i.e., reusable).

Additional features and aspects of the present invention will becomeapparent to those of ordinary skill in the art upon reading andunderstanding the following detailed description of the exemplaryembodiments. As will be appreciated by the skilled artisan, furtherembodiments of the invention are possible without departing from thescope and spirit of the invention. Accordingly, the drawings andassociated descriptions are to be regarded as illustrative and notrestrictive in nature.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying Figures, which are incorporated into and form a part ofthe specification, illustrate one or more exemplary embodiments of theinvention and, together with the general description given above anddetailed description given below, serve to explain the principles of theinvention, and wherein:

FIG. 1 is a photograph of exemplary polymer scaffolds of various shapesand sizes, in accordance with the present invention;

FIG. 2 is a photograph of exemplary polymer scaffolds of various shapesand sizes placed inside of a disposable bag bioreactor;

FIG. 3 is a photograph of randomly oriented polymer fibers depositedonto the surface of a disposable bag bioreactor;

FIG. 4 is a photograph of aligned polymer fibers deposited onto thesurface of a disposable bag bioreactor;

FIG. 5 is a photograph of dispersed or free-floating fibers in solution;

FIG. 6 is a light microscope image of dispersed polymer fiber showingsignificantly more spacing between fibers than when in a consolidatedmat or other structure;

FIG. 7 is a data graph illustrating the proliferation of humanadipocyte-derived stem cells on different types of nanofibers, inaccordance with the present invention;

FIG. 8 is a bar graph illustrating increased cell growth/expansion onthe polymer fibers of the present invention;

FIGS. 9-12 are bar graphs presenting human induced pluripotent stem cell(iPSs) and embryonic stem cell (ECs) data;

FIGS. 13-14 are bar graphs that illustrate enhanced cell growth onnanofiber scaffolds made from a blend of PET and PU compared with plainPET and plain PU using human mesenchymal stem cells; and

FIG. 15 is a photograph of a polymer fiber having a core/shellconstruction.

DETAILED DESCRIPTION OF THE INVENTION

Exemplary embodiments of the present invention are now described withreference to the Figures. Although the following detailed descriptioncontains many specifics for the purposes of illustration, a person ofordinary skill in the art will appreciate that many variations andalterations to the following details are within the scope of theinvention. Accordingly, the following embodiments of the invention areset forth without any loss of generality to, and without imposinglimitations upon, the claimed invention. With general reference to FIGS.1-15, one or more specific embodiments of this invention shall now bedescribed in greater detail.

In accordance with this invention, the process of electrospinning isdriven by the application of a high voltage, typically between 0 and 30kV, to a droplet of a polymer solution or melt at a flow rate between 0and 50 ml/h to create a condition of charge separation between twoelectrodes and within the polymer solution to produce a polymer jet. Atypical polymer solution includes a polymer such as polycaprolactone,polystyrene, or polyethersulfone and a solvent such as1,1,1,3,3,3-Hexafluoro-2-propanol, N,N-Dimethylformamide, acetone, ortetrahydrofuran in a concentration range of 1-50 wt %. As the jet ofpolymer solution travels toward the electrode it is elongated into smalldiameter fibers typically in the range of 0.1-30 μm.

In preparing an exemplary scaffold, a polymer nanofiber precursorsolution is prepared by dissolving 9 wt % polyethylene terephthalate(PET) (Indorama Ventures) in a mixture of nine parts1,1,1,3,3,3-hexafluoroisopropanol (HFIP) and one part trifluoroaceticacid. The solution is heated to 60° C. followed by continuous stirringto dissolve the PET. The solution is cooled to room temperature andplaced in a syringe (e.g., 60 cc) with a blunt tip needle (e.g., 20gauge). The nanofibers are formed by electrospinning using a highvoltage DC power supply (Glassman High Voltage, Inc., High Bridge, N.J.)set to 1 kV-40 kV (e.g., +15 kV) positive or negative polarity, a 5-30cm (e.g., 15 cm) tip-to-substrate distance, and a 1 μl/hr to 100 mL/hr(e.g., 10 ml/hr) flow rate. It is possible to use a needle arrayincluding a large number of needles (e.g., >1000) to increase systemoutput. The scaffold may be placed in a vacuum overnight and heated toensure removal of residual solvent (typically less than 10 ppm) andtreated using radio frequency gas plasma or corona for one second to oneminute to make the fibers more hydrophilic and promote cell attachmentthereto.

In accordance with this invention, it is possible to produce nanometeror micrometer sized fibers from a variety of synthetic and naturalpolymers. Suitable synthetic polymers include polycaprolactone (PCL),polyethylene terephthalate (PET), polystyrene (PS), polylactic acid(PLA), polyglycolic acid (PGA), polyurethane (PU), polyethersulfone,polyamide, Eudragit® (a polymerization of acrylic and methacrylic acidsor their esters), polyetherketoneketone (PEKK), polyglycerol sebacate(PGS), polyhydroxybutyrate (PHB), trimethylene carbonate (TMC) and/orcombinations thereof and/or derivatives thereof. Suitable naturalpolymers include gelatin, collagen, chitosan, fibrinogen, hyaluronicacid, cellulose, and/or combinations thereof and/or derivatives thereof.Suitable solvents may include acetone, dimethylformamide,trifluoroacetic acid, hexafluoroisopropanol, acetic acid,dimethylacetamide, chloroform, dichloromethane, water, ionic compounds,or combinations thereof. By predetermining the optimum fiber materialand fiber diameter for each cell type or application, higher rates ofcellular expansion, while maintaining a larger percentage of pluripotentcells, can be achieved as compared to other technologies.

The polymer fibers of this invention may include non-resorbablematerials such as polyethylene, terephthalate, silicone, polyurethane,polycarbonate, polyether-ketoneketone, polyethersulfone, polyamide,polystyrene, Eudragit®, polyethylene terephthalate, polypropylene, orcombinations thereof. and/or resorbable materials such aspolycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid (PGA),polyethylene terephthalate (PET) polypropylene (PP), or combinationsthereof, and/or materials that may be preferentially dissolved bychanges in pH, temperature or the addition of reagents to facilitatecell harvesting. Eudragit® is a pH sensitive polymer andPoly(N-isopropylacrylamide) is a thermo-responsive polymer thatfacilitates cell release from the fibers. The polymer fibers may becoated or otherwise treated with at least one compound that is operativeto promote cellular attachment to the scaffold or to prohibit unwantedcell differentiation, and those compounds may include proteins,peptides, cytokines, small molecules (e.g., drugs) or combinationsthereof. The polymer fibers of this invention may also include acore/shell construction which may be coated with a compound thatpromotes cell release therefrom.

The fiber scaffolds of the present invention may be used withbioreactors of different sizes and shapes, as well as those that aredisposable or permanent (i.e., reusable). For the purpose ofincorporating the fiber scaffolds of this invention into suchbioreactors while still facilitating easy cell removal, fiber scaffoldsof various sizes, shapes, and porosities may be utilized (see FIGS.1-2). The polymer fibers in these scaffolds may be randomly arrangedrelative to one another or may be aligned with one another (see FIGS.3-4). Depending on bioreactor geometry, the fiber scaffolds of thisinvention may be adhered to the bioreactor walls or other surfaces ormay be dispersed, individual fibers that are free-floating in the cellculture media contained in a bioreactor (see FIGS. 5-6). These variousfiber structures may be placed into the bioreactor at a manufacturingfacility, sealed, sterilized, and then shipped to the customer.Alternately, these fiber structures may be sold separately and placed inthe bioreactor by a person performing cell culture. To adhere polymerfiber to bioreactor bags, fiber is deposited directly onto the bagsurface by placing a negatively charged substrate behind the bag orplacing an anti-static bar behind the bag. This technique permitsuniform deposition of the positively charged fibers onto the bagsurface. Alternatively, the fibers may be attached to the bioreactorwalls with adhesives, heat, laser welding, ultrasonic welding, or othermethods.

In some embodiments of this invention, the fiber scaffold has beenmanufactured as a sheet of polymer fibers and then cut into pieces of atleast one predetermined size prior to placement in the bioreactor.Cutting may be accomplished with scissors, a knife, or by tearing thepolymer fiber sheet apart to form individual scaffolds of various sizessuch as, for example, about 1 mm³ fiber scaffolds to about 1 cm³ fiberscaffolds. For embodiments that utilize dispersed fibers, a tissuehomogenizer may be used to chop and shred polymer fiber sheets, whichthen allows the fibers to then be fully dispersed in liquid.

FIG. 7 is a data graph illustrating the proliferation of humanadipose-derived stem cells (hADSCs) on different types of nanofibers.Multiple replicates of hADSCs were seeded in 24-well plates containingfive different randomly oriented three-dimensional nanofiber matrices.Seven days after initial seeding, the cells were trypsinized andre-suspended in culture media. Cell enumeration and viability wasdetermined by trypan blue exclusion. Fold increases of hADSCs grown onthree-dimensional nanofiber matrices relative to control cells grown onstandard two-dimensional tissue culture polystyrene (TCPS) arequantified in FIG. 7. The data indicates that each three-dimensionalnanofiber matrix supports the growth of hADSCs and results in asignificant increase in expansion rates of cells compared to twodimensional TCPS.

FIG. 8 is a bar graph that illustrates increased cell growth/expansionon the polymer fibers of the present invention. With regard to the dataappearing in FIG. 8, multiple replicates of human mesenchymal stem cells(hMSCs) were seeded in 24-well plates containing five different randomlyoriented three-dimensional fiber matrices. Seven days after initialseeding, cells were trypsinized and resuspended in culture media. Cellenumeration and viability was determined by trypan blue exclusion. Foldincreases of hMSCs grown on three dimensional matrices relative tocontrol cells grown on standard 2D tissue culture polystyrene (TCPS) areshown in FIG. 8. The data indicate that each three-dimensional fibermatrix supports the growth of hMSCs and demonstrates a significantincrease in the expansion rates of cells cultured in three dimensionscompared to two dimensional TCPS.

FIGS. 9-12 are bar graphs presenting human induced pluripotent stem cell(iPSs) and embryonic stem cell (ECs) data. Embryonic stem cellstypically need feeder cells or collagen on which to grow and the data inthese Figures demonstrates nearly the same growth, but using a cleansynthetic fiber scaffold. This feature of the present invention isimportant for cost reduction and translation to clinical therapies dueto the superior control of the synthetic surface. FIGS. 13-14 are bargraphs that illustrate enhanced cell growth on nanofiber scaffolds madefrom a blend of PET and PU compared with just PET and/or just PU usinghuman MSCs. FIG. 15 is a photograph of a polymer fiber that iscompatible with the present invention, wherein the fiber has acore/shell construction.

While the present invention has been illustrated by the description ofexemplary embodiments thereof, and while the embodiments have beendescribed in certain detail, it is not the intention of the Applicant torestrict or in any way limit the scope of the appended claims to suchdetail. Additional advantages and modifications will readily appear tothose skilled in the art. Therefore, the invention in its broaderaspects is not limited to any of the specific details, representativedevices and methods, and/or illustrative examples shown and described.Accordingly, departures may be made from such details without departingfrom the spirit or scope of the applicant's general inventive concept.

1. A substrate for culturing cells, comprising at least one fiberscaffold adapted to be contained within a bioreactor, wherein the fiberscaffold further includes polymer fibers that have been created byelectrospinning, and wherein the orientation of the fibers in thescaffold relative to one another is generally parallel, random, or both.2. The substrate of claim 1, wherein the bioreactor is a disposablebioreactor.
 3. The substrate of claim 1, wherein the bioreactor is anon-disposable bioreactor.
 4. The substrate of claim 1, wherein thefiber scaffold is attached to at least one interior surface of thebioreactor.
 5. The substrate of claim 1, wherein the fiber scaffold isadapted to float freely in the bioreactor when the bioreactor is filledwith cell culture media.
 6. The substrate of claim 1, where in the fiberscaffold has been manufactured as a sheet of polymer fibers, and whereinthe sheet of polymer fibers has been cut into pieces of at least onepredetermined size prior to placement in the bioreactor, and wherein theat least one predetermined size ranges from about 1 mm3 to about 1 cm3.7. The substrate of claim 1, wherein the fiber scaffold further includesindependent fibers that are adapted to be dispersed in cell culturemedia used in a bioreactor.
 8. The substrate of claim 1, wherein thepolymer fibers have been coated or otherwise treated with at leastcompound that is operative to promote cellular attachment to thescaffold, and wherein the at least one compound further includesproteins, peptides, cytokines, small molecules, or combinations thereof.9. The substrate of claim 1, wherein the polymer fibers further includesynthetic polymers, and wherein the synthetic polymers further includepolycaprolactone, polyethylene terephthalate, polystyrene, polylacticacid, polyglycolic acid, polyurethane, polyethersulfone, polyamide,polyetherketoneketone, EUDRAGIT, polyglycerol sebacate,polyhydroxybutyrate, trimethylene carbonate, and combinations thereof orderivatives thereof.
 10. The substrate of claim 1, wherein the polymerfibers further include natural polymers, and wherein the naturalpolymers further include gelatin, collagen, fibrinogen, hyaluronic acid,cellulose, and combinations thereof or derivatives thereof.
 11. Thesubstrate of claim 1, wherein the polymer fibers further includenon-resorbable materials, and wherein the non-resorbable materialsfurther include polyethylene, terephthalate, silicone, polyurethane,polycarbonate, polyether-ketoneketone, polyethersulfone, polyamide,polystyrene, EUDRAGIT, polyethylene terephthalate, polypropylene, orcombinations thereof.
 12. The substrate of claim 1, wherein the polymerfibers further include resorbable materials, and wherein the resorbablematerials further include polycaprolactone, polylactic acid,polyglycolic acid, trimethylene carbonate, polyhydroxylbutyrate, orcombinations thereof.
 13. The substrate of claim 1, wherein the polymerfibers further include materials that may be preferentially dissolved bychanging pH, changing temperature, or by the addition of reagents thatfacilitate the harvesting of cells from the fibers.
 14. A system forculturing cells, comprising: (i) at least one fiber scaffold adapted tobe contained within a bioreactor, wherein the fiber scaffold furtherincludes polymer fibers that have been created by electrospinning, andwherein the orientation of the fibers in the scaffold relative to oneanother is generally parallel, random, or both; and (ii) a bioreactor.15. The system of claim 14, wherein the bioreactor is a disposablebioreactor.
 16. The system of claim 14, wherein the bioreactor is anon-disposable bioreactor.
 17. The system of claim 14, wherein the fiberscaffold is attached to at least one interior surface of the bioreactor.18. The system of claim 14, wherein the fiber scaffold is adapted tofloat freely in the bioreactor when the bioreactor is filled with cellculture media.
 19. The system of claim 14, where in the fiber scaffoldhas been manufactured as a sheet of polymer fibers, and wherein thesheet of polymer fibers has been cut into pieces of at least onepredetermined size prior to placement in the bioreactor.
 20. The systemof claim 14, wherein the fiber scaffold further includes independentfibers that are adapted to be dispersed in cell culture media used in abioreactor.
 21. The system of claim 14, wherein the polymer fibers havebeen coated or otherwise treated with at least compound that isoperative to promote cellular attachment to the scaffold, and whereinthe at least one compound further includes proteins, peptides,cytokines, small molecules, or combinations thereof.
 22. The system ofclaim 14, wherein the polymer fibers further include synthetic polymers,and wherein the synthetic polymers further include polycaprolactone,polyethylene terephthalate, polystyrene, polylactic acid, polyglycolicacid, polyurethane, polyethersulfone, polyamide, polyetherketoneketone,EUDRAGIT, polyglycerol sebacate, polyhydroxybutyrate, trimethylenecarbonate, and combinations thereof or derivatives thereof.
 23. Thesystem of claim 14, wherein the polymer fibers further include naturalpolymers, and wherein the natural polymers further include gelatin,collagen, fibrinogen, hyaluronic acid, or cellulose, and combinationsthereof or derivatives thereof.
 24. The system of claim 14, wherein thepolymer fibers further include non-resorbable materials, and wherein thenon-resorbable materials further include polyethylene, terephthalate,silicone, polyurethane, polycarbonate, polyether-ketoneketone,polyethersulfone, polyamide, polystyrene, EUDRAGIT, polyethyleneterephthalate, polypropylene, or combinations thereof.
 25. The system ofclaim 14, wherein the polymer fibers further include resorbablematerials, and wherein the resorbable materials further includepolycaprolactone, polylactic acid, polyglycolic acid,polyhydroxybutyrate, trimethylene carbonate, or combinations thereof.26. The system of claim 14, wherein the polymer fibers further includematerials that may be preferentially dissolved by changing pH, changingtemperature, or by the addition of reagents that facilitate theharvesting of cells from the fibers.
 27. The system of claim 14, whereinthe polymer fibers further include a core/shell construction.